Phospholipid derivatized with PEG bifunctional linker and liposome containing it

ABSTRACT

A phospholipid derivative of the following formula (I): ##STR1## wherein A is a residue of a phospholipid having a phosphatidylethanolamine moiety, and B is a linking group having a polyalkylene glycol moiety.

The present invention relates to a novel phospholipid derivative and aliposome containing it. Particularly, it relates to a novel phospholipidderivative having a polyalkylene glycol moiety having a maleimide group.

A liposome being a lipid vesicle is capable of containing manysubstances irrespective of whether such substances are water-soluble orhydrophobic and thus is expected to be a prospective carrier especiallyfor a drug delivery system (DDS).

In recent years, for the purpose of imparting a functionality to theliposome in addition to the inherent properties thereof, an attempt hasbeen made to bond (or introduce) a functional compound such as aprotein, a peptide, a sugar or a hydrophilic polymer to the liposomesurface.

On the other hand, many studies have been made to overcome generaldrawbacks of a liposome, such as agglomeration and non-specific captureby a reticuloendothelial system organ such as liver or spleen, and ithas been found effective to bond a polyethylene glycol to the liposome(Japanese Unexamined Patent Publications No. 249717/1989 and No.149512/1990, FEBS letters, 268,235 (1990)).

Further, for the purpose of imparting the function of protein and theproperties of polyethylene glycol, methods have been proposed to producea liposome which contains both a protein and a polyethylene glycol (BBA,1062:142 (1991), and Japanese Unexamined Patent Publication No.346918/1992).

The former is a method which comprises solubilizing a lipid-modifiedantibody, a phospholipid, cholesterol and a lipid derivative ofpolyethylene glycol by means of a surfactant, followed by removing thesurfactant by dialysis, to obtain a liposome. However, it is difficultto remove the surfactant completely, and the product will hardly beuseful as a drug. Further, it is necessary to carry out the dialysis fora long period of time at a temperature higher than the phase transfertemperature of the lipid, whereby a useful lipid is rather limitedespecially when an unstable protein is employed for the production.

The latter is a method which comprises preparing a liposome containingmaleimide groups, then bonding a protein having thiol groups and furtherbonding a polyethylene glycol having thiol groups to excess maleimidegroups. While the protein can be bonded under a mild condition, thismethod has a problem that the production process is cumbersome, since itinvolves a two step reaction, and it is difficult to independentlycontrol the amount of each component to be bonded.

The present inventors have conducted a study to solve such problems ofconventional liposomes containing both a protein and a polyalkyleneglycol and as a result, have found it possible to readily produce aliposome containing both a protein and a polyalkylene glycol by using,as a liposome-constituting component, a phospholipid derivative having amaleimide group at one end of a polyalkylene glycol and aphosphatidylethanolamine moiety at the other end. Further, it has beensurprisingly found that the liposome containing such a phospholipidderivative can readily be micronized during the process for micronizingthe liposome. The present invention has been accomplished on the basisof these discoveries.

Namely, the present invention provides a phospholipid derivative of thefollowing formula (I) and a liposome containing such a derivative:##STR2## wherein A is a residue of a phospholipid having aphosphatidylethanolamine moiety, and B is a linking group having apolyalkylene glycol moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the ¹ H-NMR chart of M-PEG-PE obtained inExample 1.

FIG. 2 is a graph showing the results of measurement of the particlesize distribution of the M-PEG-PE-containing liposome prepared inExample 3 by a dynamic light scattering method.

FIG. 3 is a graph showing the results of measurement of the particlesize distribution of the liposome containing no M-PEG-PE, prepared inComparative Example, by a dynamic light scattering method.

FIG. 4 is a photograph showing the reactivity of the antibody-bondedliposome (immuno liposome) prepared in Example 4 to human stomach cancercells MKN45. The observation was made by a fluorescent microscope usinga fluorescent lipid integrated to the liposome, as a marker.

FIG. 5 is a photograph showing the reactivity of the non-antibody-bondedliposome prepared in Comparative Example to human stomach cancer cellsMKN45. The observation was made by a fluorescent microscope using afluorescent lipid integrated to the liposome, as a marker.

FIG. 6 is a photograph showing the luminous field image of FIG. 5.

Now, the present invention will be described in detail.

The phospholipid derivative of the present invention is represented bythe above formula (I). The residue of a phospholipid havingphosphatidylethanolamine moiety for A, is not particularly limited solong as it is the one having a phosphatidylethanolamino group. However,it may preferably be a group of the following formula (II): ##STR3##wherein each of R¹ and R² which are independent of each other, is a C₁₁-C₁₉ alkyl group such as a lauroyl group, a myristoyl group, a palmitoylgroup or a stearoyl group, or a C₁₁ -C₁₉ alkenyl group such as an oleylgroup, and D is a single bond, or a C₁ -C₁₁ linking group such as--C═(NH₂ ⁺)(CH₂)₀₋₁₀ --, --CO(CH₂)₀₋₁₀ -- or --CH₂ (CH₂)₀₋₁₀ --.

The linking group having a polyalkylene glycol moiety for B, is notparticularly limited so long as it is the one having a polyalkyleneglycol unit such as polyethylene glycol, polypropylene glycol,polytetramethylene glycol or polyhexamethylene glycol. However, it maypreferably be a linking group of the following formula (III):

    -J-PEG-L-                                                  (III)

wherein each of J and L which are independent of each other, is a singlebond, or a C₁ -C₁₁ linking group such as --CO(CH₂)₀₋₁₀ --, --(CH₂)₀₋₁₀CO--, --NHCO(CH₂)₀₋₁₀ --, --(CH₂)₀₋₁₀ CONH--, or --CH₂ (CH₂)₀₋₁₀ --, andPEG is a polyethylene glycol residue such as --(CH₂ CH₂ O )₁₁₋₄₅₅ --.

Now, a process for producing the phospholipid derivative of the presentinvention will be described.

The phospholipid derivative of the present invention can be produced,for example, by reacting a lipid having a thiol group with apolyalkylene glycol having two maleimide groups. ##STR4## In the aboveformulas, A and B are as defined above.

Now, the process will be described sequentially in detail.

1. Preparation of the phospholipid having a thiol group (hereinaftersometimes referred to simply as "the thiol-modified phospholipid")

The thiol-modified phospholipid can be obtained by introducing a thiolgroup to phosphatidylethanolamine by means of a known aminogroup-modifying reaction. Namely, it can be produced by dissolving aTraunt reagent such as iminothiolane or a mercapto alkyl imidate andphosphatidylethanolamine in an organic solvent such as chloroform orchloroform/methanol (1/1 to 10/1) in the presence of a basic compoundsuch as triethylamine or pyridine and reacting them at a temperature offrom 20 to 40° C. under a condition of an inert gas such as nitrogen gasor argon gas. Otherwise, it can be prepared by means of a thiolcarboxylic acid disclosed in Japanese Unexamined Patent Publication No.72067/1989. Or, it may be synthesized by bonding a compound inherentlycontaining a sulfur atom, such asN-succinimidyl-3-(2-pyridyldithio)propionate, tophosphatidylethanolamine, followed by reducing with a large excessamount of a reducing agent such as 2-mercaptoethanol.

The phosphatidylethanolamine to be used here, i.e. the phospholipidhaving a phosphatidylethanolamine moiety, for A in the above formula(I), is not particularly limited and may, for example, be naturalphosphatidylethanolamine derived from e.g. yolk, dioleylphosphatidylethanolamine, dimyristoyl phosphatidylethanolamine,dipalmitoyl phosphatidylethanolamine or distearoylphosphatidylethanolamine, preferably dipalmitoylphosphatidylethanolamine.

2. Preparation of the polyalkylene glycol having two maleimide groups(hereinafter sometimes referred to

simply as "the maleimide PAG") The process will be described in detailwith respect to a case wherein the polyalkylene glycol moiety ispolyethylene glycol (hereinafter sometimes referred to simply as "PEG").

The polyethylene glycol derivative having two maleimide groups can beobtained, for example, by dissolving diaminopolyethylene glycol(available from Nippon Oil and Fats or Sigma) and at least a two-foldmola amount of a maleimide-modifying reagent such asN-(ε-maleimidocaproyloxy)succinimide in an organic solvent such aschloroform dehydrated by e.g. molecular sieve, in the presence of abasic compound such as triethylamine or pyridine and reacting them at atemperature of from 20° to 40° C. under an atmosphere of an inert gassuch as nitrogen gas or argon gas.

In addition to N-(ε-maleimidocaproyloxy)succinimide, themaleimide-modifying reagent may, for example, be N-succinimidyl4-(p-maleimidophenyl)butyrate, N-succinimidyl4-(p-maleimidophenyl)propionate or N-(γ-maleimidobutyryloxy)succinimide,which is commonly used for the preparation of a maleimide derivative ofan amino group.

The average molecular weight of the PEG moiety is usually from 500 to20000, preferably from 2000 to 10000, more preferably from 2000 to 5000.As such PEG, SUNBRIGHT® VFM5001 (Nippon Oil and Fats) orPolyoxyethylenebisamine (Sigma) may specifically be mentioned.

3. Preparation of the compound of the present invention (hereinaftersometimes referred to simply as "M-PAG-PE")

M-PAG-PE can be prepared by adding and reacting the thiol-modifiedphospholipid dissolved in an organic solvent such as chloroform orchloroform/methanol (1/1 to 10/1) to the dimaleimide PAG dissolved inthe same solvent in the presence of a basic compound such astriethylamine or pyridine at a temperature of from 0° to 40° C. under anatmosphere of an inert gas such as nitrogen gas or argon gas. Here, thethiol-modified phospholipid is gradually added, and its total amount isusually at most an equimolar amount, preferably at most 1/2 mol of themaleimide PAG.

Then, hexane or petroleum ether is added to the reaction solution ofM-PAG-PE to precipitate a PAG-containing compound. The precipitatecollected by filtration is dried and then dissolved in distilled waterat a concentration not lower than the concentration where M-PAG-PEitself forms particles (micell) in the aqueous solution. Insolublematters are filtered off, and while maintaining the concentration at theabove specified level, the solution is subjected to molecular weightfractionation by e.g. an ultrafiltration method, a dialysis method or agel filtration method to obtain M-PAG-PE. At that time, impurities suchas unreacted (excess) dimaleimide PAG are removed. Further, ifnecessary, purification by silica gel column chromatography may furtherbe carried out.

The phospholipid derivative of the present invention thus obtained canbe converted to a liposome by a conventional method. The liposome may beformed by a conventional lipid component such as phosphatidylcholine orcholesterol, and M-PAG-PE, or may be formed by mixing such aconventional lipid component, M-PAG-PE and another lipid derivativehaving a polyethylene glycol moiety as disclosed in e.g. JapaneseUnexamined Patent Publication No. 249717/1989 or No. 149512/1990 or FEBSletters, 268,235 (1990).

The phosphatidylcholine useful as the lipid component is notparticularly limited and may, for example, be naturalphosphatidylcholine derived from e.g. yolk, dioleyl phosphatidylcholine,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine ordistearyl phosphatidylcholine.

The respective components are used in such proportions that per mol ofthe phosphatidylcholine, the cholesterol is used in an amount of from0.3 to 1 mol, preferably from 0.4 to 0.6 mol, and M-PAG-PE is used in anamount of from 0.001 to 0.4 mol, preferably from 0.02 to 0.1 mol.Further, when another lipid derivative having a polyethylene glycolmoiety is incorporated, such another lipid derivative is used in anamount of not more than 0.4 mol per mol of the phosphatidylcholine.

Then, the lipid mixture having the solvent removed, may, for example, behydrated and emulsified by a homogenizer, followed by freezing-thawingto obtain a multilamella liposome (MLV). Further, it may be subjected tosuper sonic treatment, high speed homogenizing or press-filtration witha membrane having uniform pores (Hope M. J. et al, Biochimica etBiophysica Acta, 812, 55 (1985)) to obtain a single lamella liposome(SUV) and to adjust the particle size to a proper level. Here, apreferred particle size is from 20 to 300 nm, more preferably from 30 to200 nm.

Various drugs may be loaded into such a liposome. As such a drug, anantitumor drug such as adriamycin, daunomycin, mitomycin, cisplatin,vincristine, epirubicin, methotrexate, 5FU (5-fluorouracil) oracracinomycin, an aminoglucoside such as gentamicin, a β-lactamantibiotic such as sulpenisillin, a toxin such as ricin A or diphtheriatoxin, or an antisense RNA against human immunodeficiency virus (HIV),hepatitis B virus, hepatitis C virus or ras gene, may, for example, bementioned.

Loading of the drug into the liposome can be conducted by hydrating thelipid with an aqueous drug solution in the case of a water-soluble drug,or by mixing the drug and the lipid in a volatile organic solvent,followed by distilling the solvent off and hydrating the mixture of thedrug and the lipid to incorporate the drug into the liposome, in thecase of a lipophilic drug.

Further, for the purpose of imparting a functionality to liposome, it ispreferred to bond (introduce) a protein, a peptide, a saccharide, ahydrophilic polymer, etc. to the surface of the liposome, as mentionedabove. In the present invention, it is preferred to bond (introduce)various proteins including antibodies or growth factories such asfibroblast growth factor (FGF) and epitheliocyte growth factor (EGF).Particularly preferred are antibodies. The antibodies are those (such asIgA, IgG and IgM) which are reactive with the tissues, cells, bacteriaor virus to be treated. For example, polyclonal antibodies of variousanimals, a mouse monoclonal antibody, a human-mouse chimeric antibodyand a human monoclonal antibody may be employed.

To bond such a protein to the liposome, the double bond of the maleimidegroup of the liposome and the thiol group of the protein can beutilized. Introduction of a thiol group to the protein can be conductedby a method wherein a compound is employed which is commonly used forthiol-modification of a protein and which is reactive with an aminogroup of the protein, such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson, J. etal., Biochem. J. 173, 723 (1978)) or iminothiolane, mercaptoalkylimidate(Traut, R. R. et al., Biochemistory, 12, 3266 (1973)). In the case wherethe protein is an antibody, a method may be employed wherein endogenousdithiol groups in the cystine residues are reduced to thiol groups.

When IgG is employed among antibodies, it may be subjected to F(ab')₂modification by treatment with an enzyme such as pepsin, followed byreduction with e.g. dithiothreitol to obtain Fab', whereupon thiolgroups formed in Fab' are subjected to the bonding reaction with theliposome (Martin, F. J. et al., Biochemistory, ,20, 4229 (1981)). In thecase of IgM, J-chain may be reduced under a mild condition in accordancewith a method of Miller et al. (J. Biol. Chem. 257, 286 (1965)),whereupon thiol groups of Fc moiety of IgMs thereby obtained, aresubjected to the bonding reaction with the liposome.

The bonding of the liposome with such a protein can be accomplished byreacting them in a neutral buffer solution (pH 6.0 to 7.5) for from 2 to16 hours.

To use the liposome of the present invention as a pharmaceuticalcomposition, it may be administered by e.g. intravascular administrationor local administration such as intravesical or intraperitonealadministration against various diseases. The dose may optionally beadjusted depending upon the pharmacological activities, the type of thedrug, etc.

Now, the present invention will be described in further detail withreference to Examples. However, it should be understood that the presentinvention is by no means restricted to such specific Examples.

EXAMPLE 1 Preparation of M-PEG-PE

Preparation of thiol-modified phospholipid

21.8 mg of iminothiolane (manufactured by Sigma Company) was added to100 mg of dipalmitoyl phosphatidylethanolamine dissolved in 11 ml of amixture of chloroform/methanol (6/5). Then, 50 μl of triethylamine wasfurther added thereto, and the mixture was stirred and reacted at roomtemperature in nitrogen gas until the ninhydrin reaction becamenegative. Formation of the thiol-modified phospholipid was confirmed byadding fluoresce maleimide (manufactured by Funakoshi) to a part of thereaction solution, reacting them at room temperature for 40 minutes andobserving a fresh yellow fluorescent spot on a thin layerchromatography.

Preparation of dimaleimide PEG

810 mg of diamino polyethylene glycol (SUNBRIGHT® VFM5001 (manufacturedby Nippon Oil and Fats); average molecular weight: 5000) was dissolvedin 5 ml of chloroform dehydrated by a molecular sieve, and 100 mg ofN-(ε-maleimidocaproyloxy)succinimide and 50 μl of triethyamine wereadded thereto. The mixture was stirred and reacted at room temperaturein nitrogen gas. Formation of dimaleimide PEG was ascertained byconfirming that the ninhydrin reaction became negative.

Bonding and purification

To 4 ml of the above dimaleimide PEG solution diluted to 30 ml withchloroform, 10 ml of the above thiolmodified phospholipidsolution wasgradually added, and the mixture was stirred at room temperature for 3hours under a nitrogen gas condition. To 20 ml of the reaction solutionthus obtained, an adequate amount of hexane was added to form aprecipitate containing M-PEG-PE. The precipitate was vacuum-dried,whereupon the weight was 330 mg. It was dissolved in 10 ml of distilledwater, and insoluble substances were removed by centrifugal separationof 16000× g for 5 minutes. Further, the same amount of distilled waterwas added, and concentration-filtration was repeated by means ofCENTRICON 100 (manufactured by Amicon) equipped with an ultra filtermembrane with a molecule weight fraction of 100 k. By this purificationprocess, PEG not combined with the lipid derivative, was filtered off.

Measurement by NMR

As shown by the ¹ H-NMR chart of the desired product in FIG. 1, a signalof 1.54 ppm of the alkyl chain proton of the phospholipid, a signal of3.57 ppm attributable to --CH₂ CH₂ O-- of PEG and a signal of 6.61 ppmattributable to the double bond of the maleimide, were confirmed.

REFERENCE EXAMPLE 1

As shown in Example 1, the phospholipid derivative of the presentinvention was considered to behave as a molecular aggregate in theaqueous solution. Therefore, the form of M-PEG-PE in the aqueoussolution was studied.

M-PEG-PE dissolved in distilled water at a concentration of 0.5 mg/ml,was measured by a dynamic light scattering method (ELS-800, manufacturedby Otsuka Denshi), whereby particles of 24.8 nm (g (GAMMA) distribution)were observed.

EXAMPLE 2

Measurement of maleimide

The maleimide content of M-PEG-PE obtained in Example 1 was determinedby adding cysteine to the aqueous solution and quantitatively analyzingthe SH amount consumed by the maleimide by means of 4,4'-dithiopyridine.Namely, an aqueous solution containing 1.1 mg/ml of M-PEG-PE was mixedin the following proportion, and the mixture was reacted at roomtemperature for 30 minutes.

    ______________________________________    M-PEG-PE             100 μl    1 mM EDTA-containing 0.1 M                         800 μl    phosphate buffer (pH 6.0)    0.5 mM cysteine      100 μl    ______________________________________

After the reaction, 40 μl of 5 mM 4,4'-dithiopyridine was added to 960μl of the above solution, and the maleimide content was determined bycomparing the change in the absorbance at 324 nm by the color developedby the reaction with the remaining cysteine, with that of the controlsample (a mole absorbance coefficient of the developed color of 19800/Mwas employed).

As a result, a maleimide amount of 0.17 μmol per mg of M-PEG-PE wasdetected.

EXAMPLE 3

Preparation of liposome containing M-PEG-PE

50 mg of dipalmitoyl phosphatidylcholine, 14.6 mg of cholesterol, 10.7mg of M-PEG-PE and 0.16 mg of FITC-DPPE(1,2-Dihecadecanoyl-sn-glycero-3-phospho[N-(5-fluoresceinthiocarbamoyl)]ethanolamine,manufactured by Sigma) as a fat-solble marker, were dissolved uniformlyin chloroform, and the solvent was removed by an evaporator to form alipid film. Further, the film was dried under reduced pressure by avacuum pump for 2.5 hours, and then a 0.1M phosphate buffer solution (pH6.0) and 1 ml of 1 mM EDTA were added thereto, the mixture was stirredand hydrated by a voltex mixer. Then, freezing-thawing was repeated fivetimes by means of liquid nitrogen and warm bath of 65° C., to obtain amilky white multilamella liposome (MLV).

Then, 0.5 ml of the above MLV was adjusted to 1 ml with the above buffersolution and subjected to supersonic treatment for 10 minutes by aProbe-type sonicater (Sonifier 450, manufacture by Branson Company) atan output of 20%, to obtain a transparent small diameter liposome (SUVsolution). FIG. 2 shows the particle size distribution of the liposomethus obtained, as measured by a dynamic light scattering method, it isshown that there was no inclusion of particles having a diameter of morethan 1 μm. COMPARATIVE EXAMPLE

MLV was prepared in the same manner as in Example 3 except that thelipid composition did not contain M-PEG-PE. Supersonic treatment for 10minutes was repeated twice in the same manner, and then the particlesize distribution was measured by a dynamic light scattering method. Theresults are shown in FIG. 3. Particles having a diameter of more than 1μm were included, thus indicating that no adequate micronization wasaccomplished.

EXAMPLE 4

Bonding of an antibody to the M-PEG-PE-containing liposome

To an antitumor human monoclonal antibody (IgG; GAH disclosed in EP0520499 A), 1/40 mol amount of pepsine (Cooper Biomedical) in 0.1Macetic acid buffer solution (pH 4.0), was added, and the mixture wasreacted at 37° C. over night for digestion to obtain F(ab')₂.

Further, by chromatography separation with a cation exchange resin (MonoS, manufactured by Pharmacia), F(ab')₂ was isolated. The preparation wasconducted by a linear gradient from 0M to 1.0M NaCl in a 0.1M aceticacid buffer solution (pH 4.0), to obtain F(ab')₂.

The buffer solution was changed to a 50 mM phosphate buffer solution (pH7.5) and 1 mM EDTA in a PD-10 column (manufacture by Pharmacia) toobtain a solution having an antibody concentration of 2.4 mg/ml. Then,iminothiolane (3 mg/ml) was added thereto in an amount corresponding to4 mol times of the antibody, and the mixture was reacted at 37° C. forone hour. Unreacted iminothiolane was removed by demineralization by aPD-10 column equilibrated with a 0.1M phosphate buffer solution (pH 6.0)and 1 mM EDTA, to obtain 1.9 mg/ml of a thiolmodified antibody.

To 0.7 ml of this antibody, 0.42 ml of the SUV liposome obtained inExample 3, was added, and the mixture was shaked at room temperatureover night to obtain an immunoliposome.

EXAMPLE 5

Reactivity to the target cells

Using human gastric cancer cell line MKN 45 (having a reactivity withthe antibody used in Example 4) which was cultured on a slide chamber(LAB-TEK® chamber, manufactured by Nunc, Inc.) and fixed byparaformaldehyde, the reactivity of the immunoliposome prepared inExample 4, was confirmed. A reaction solution comprising 50 μl of theliposome solution, 150 μl of a human serum and 50 μl of a 10 mMphosphate buffer solution (pH 7.4) and 0.15M NaCl , was added to thecells, and the mixture was incubated at 37° C. for 30 minutes. Afterwashing with the same buffer solution, fluorescence of FITC-DPPE (asmentioned above) introduced as a marker, was observed by a fluorescencemicroscope (Confocal microscope adoptor VX100, manufactured by Newport).The respective fluorescence microscopic photographs are shown in FIG. 4(antibody-bonded liposome), FIG. 5 (non antibody-bonded liposomeprepared in Comparative Example) and FIG. 6 (Luminous field image ofFIG. 5). As compared with the liposome used as control, high reactivityof the immuno liposome of the present invention to MKN45 cells wasconfirmed.

The phospholipid derivative of the present invention is useful as aphospholipid component constituting a liposome, and the liposomeemploying the phospholipid derivative has a polyalkylene glycol moiety,whereby non-specific uptake by a recticuloendothelial system such asliver or spleen can be suppressed and at the same time the liposome canbe readily bonded to a protein having thiol moiety. Further, by means ofthe phospholipid derivative of the present invention, the liposome canreadily be micronized.

We claim:
 1. A phospholipid derivative of formula (I): ##STR5## whereinA has the following formula (II): ##STR6## wherein each of R¹ and R²,which are independent of each other, is a C₁₁ -C₁₉ alkyl group or a C₁₁-C₁₉ alkenyl group, and D is a single bond, --C═(NH₂ ⁺)(CH₂)₀₋₁₀ --,--CO(CH₂)₀₋₁₀ -- or --CH₂ (CH₂)₀₋₁₀ --, and B is a linking group havingthe following formula (III):

    J-PEG-L                                                    (III)

wherein each of J and L, which are independent of each other, is asingle bond, --CO(CH₂)₀₋₁₀ --, --(CH₂)₀₋₁₀ CO--, --NHCO(CH₂)₀₋₁₀ --,--(CH₂)₀₋₁₀ CONH-- or --CH₂ (CH₂)₀₋₁₀ --, and PEG is a polyethyleneglycol residue.
 2. A liposome comprising the phospholipid derivative ofclaim
 1. 3. The liposome according to claim 2 further comprising a drug.4. The liposome according to claim 1, wherein a protein having a thiolgroup is reacted with a double bond of a maleimide group of thephospholipid derivative.
 5. The liposome according to claim 4, whereinthe protein having a thiol group is an antibody wherein endogenousdithiol groups in cysteine residues thereof have been reduced to thiolgroups.